CN104548083B - A kind of vaccine composition and its preparation method and application - Google Patents
A kind of vaccine composition and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of vaccine composition, which includes circovurus type 2 antigen and levamisol of immune amount or derivatives thereof.The vaccine composition can increase substantially the immune efficacy of vaccine, while may be used as the diluent of the live virus antigen of freeze-drying, and the vaccine composition has stability, the indifference when measurement of its immune efficacy was just prepared with 0 day after long-time preservation.
Description
Technical field
The present invention relates to a kind of vaccine compositions, specifically, being related to a kind of circovirus vaccine composition.
Background technique
Pig circular ring virus (PCV) is a kind of the smallest animal virus found so far.Known PCV there are two serotype,
That is PCV1 and PCV2.PCV1 is the virus of non-pathogenic.PCV2 is pathogenic virus, it is that postweaning multisystemic failure is comprehensive
The main pathogen of simulator sickness (PMWS).The Clinical symptoms of PMWS has syntexis, ochrodermia, expiratory dyspnea, diarrhea, yellow subcutaneous ulcer etc..Pathology
Change visible lymph nodes of body as a whole enlargement, especially inguinal lymphadenopathy is up to 5~10 times；Lung is mainly in dispersivity, interstitial
Property pneumonia variation, hard such as rubber, surface is generally in the mottling appearance of taupe；The variation of kidney is more, it is seen that cortex and
Medullary substance is dispersed in white necrosis region not of uniform size, causes it that waxy appearance is presented due to oedema；Spleen silght enlargement；Most sick pig
Liver have different degrees of atrophy, fibrosis.
In addition to PMWS, pigskin inflammation and nephrotic syndrome (PDNS), Hypertrophic necrotizing pneumonia (PNP), porcine respiratory disease
Syndrome (PRDC), breeding difficulty, it is congenital tremble, the diseases such as enteritis are also with PCV2 infected with important association.PCV2 is relevant
Swine disease, the death rate are differed up to 10%~30%, and more serious pig farm death and culling rate when breaking out is up to 40%, are caused seriously to pig breeding industry
Economic loss.The prior art is by the subunit vaccine of the CAP albumen of inactivated virus vaccine and PCV2 preparation for preventing
PMWS, however inactivated virus vaccine cause higher cost since the cell yield of PCV is not high, and subunit vaccine has not
It is proliferated in animal body, no Exogenous Nucleic Acid, therefore does not have the advantages that infectiousness, but there is also purify at high cost ask simultaneously
Topic.The prior art needs a kind of low in cost and efficient vaccine for being directed to circovirus.
Summary of the invention
To solve the deficiencies in the prior art, the present invention provides a kind of circovirus vaccine, includes: circovurus type 2 antigen,
Levamisol or derivatives thereof, polymer and pharmaceutically acceptable carrier.
The main purpose of the present invention is to provide a kind of vaccine composition, the vaccine composition includes the annulus of immune amount
Viral 2 type antigens and levamisol or derivatives thereof.
Term " porcine circovirus 2 type antigen " refers to any composition containing at least one PCV2 antigen forms, described
Antigen inoculation pig can induce, the immune response that PCV2 infects is resisted in stimulation or enhancing.It is entirely sick that the PCV2 antigen can be PCV2
Poison, the PCV2 totivirus such as inactivated, the embedded virus for containing PCV2 immunogen amino acid sequence (such as ORF2 albumen), Ren Heqi
Its at least polypeptide of the immunogen amino acid sequence containing PCV2 or subunit or other compositions.PCV2 antigen can also include following
Any antigen of composition: such as Cimmeria company (Merial)Pfizer (Pfizer)PCV2；The porcine circovirus 2 type inactivated vaccine (LG plants) of Harbin Wei Ke biotechnology development company；Foochow
The porcine circovirus 2 type inactivated vaccine (DBN-SX07 plants) and genetic engineering subunit vaccine of Da Bei Nong Bioisystech Co., Ltd
(such as Ingelvac of Boehringer Ingelheim company (Boehringer-Ingelheim)Etc.) because of PCV2
Genome Size is 1767bp or 1768bp, and nucleotide sequence homology is higher between different strains, 90% or more, therefore
PCV2SH plants (GenBank accession number is AY686763), PCV2LG plants of (GenBank accession number is HM038034), PCV2DBN-
SX07-2(GenBank accession number is HM641752) within the present invention.
Preferably, the circovurus type 2 antigen is Porcine circovirus type 2 ORF2 protein, and the Porcine circovirus type 2 ORF2 protein is 2~300 micro-
Gram/mL, more preferable 2~60 micrograms/mL, most preferably 60 micrograms/mL.
Preferably, the circovurus type 2 antigen is the porcine circovirus 2 type totivirus antigen of inactivation, the pig annulus
Viral 2 type totivirus antigens be inactivation before >=106.0TCID50。
Totivirus antigen after circovurus type 2 antigen of the present invention, preferably subunit antigen or inactivation.As this hair
A kind of bright embodiment, totivirus antigen are SH plants of totivirus antigens, can be according to disclosed in patent application CN101240264A
Method, bacterial strain preparation, as another embodiment of the invention, antigen is Porcine circovirus type 2 ORF2 protein, can be according to patent application
Method disclosed in CN103173470A, gene source preparation.
The SH bacterial strain, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date
On March 4th, 2008, deposit number are CGMCC No.2389.
The antigenic content of circovirus includes at least 2 microgram Porcine circovirus type 2 ORF2 proteins/dosage, preferably 2~300 micrograms
Porcine circovirus type 2 ORF2 protein/dosage, more preferable 20-150 microgram Porcine circovirus type 2 ORF2 protein/dosage, more preferable 50-100 microgram PCV2ORF2
Protein/dose, more preferable 60 microgram Porcine circovirus type 2 ORF2 protein/dosage.
It is 1 × 10 that the inactivated vaccine inactivation provirus content of totivirus, which states pig circular ring virus antigen,5.0TCID50/ dosage with
On, preferably 106.0TCID50It is more than/dosage.
Preferably, levamisol derivative is levamisol hydrochloride or phosphate, described levamisol or derivatives thereof
Content be 0.1~10 milligram/mL, more preferable 1.5~3 milligrams/mL.
Term levamisol is molecular formula C11H12N2S compound, the derivative of levamisol refer to levamisol derivative
It is its hydrochloride or phosphate, is the amount of levamisol by the conversion of its molecular weight when use.
Preferably 0.1 milligram~10 milligrams/dosage of levamisol or derivative, more preferable 0.5 milligram of g~5 milligram/dosage,
More preferable 1.0 milligrams~3 milligrams/dosage, more preferable 1.5 milligrams~2 milligrams/dosage most preferably 1.5 milligrams/dosage.
Preferably, the vaccine composition further comprises polymer, and the polymer includes deae dextran, poly- second two
Alcohol, polyacrylic acid and polymethylacrylic acid, preferably polyacrylic acid, more preferable carbomer, the polymer content be 0.1~
500 milligrams/mL, more preferable 2 milligrams //mL.
Vaccine composition may also include one or more polymer, such as, such as: deae dextran, polyethylene glycol and poly- third
Olefin(e) acid and polymethylacrylic acid.This substance can be bought from market.Measurement suitable for the polymer in adjunvant composition
Certainly in the property of used polymer.Preferably, polymer of the present invention is polyacrylic acid；It is highly preferred that the polymerization
Object is the polyacrylic acid of trade name carbomer (Carbomer)；
The amount of polymer of the present invention be 0.1 milligram~500 milligrams/dosage, preferably 1 milligram~20 milligrams dosage, more
It is preferred that 5 milligrams~10 milligrams/dosage, more preferable 1 milligram~3 milligrams dosage, most preferably 2mg/ dosage.
Preferably, the vaccine composition further comprises DDA, and DDA content is 1~5000 microgram/mL, more preferable 50~
150 micrograms/mL.
As another embodiment of the invention, DDA is also contained in composition, the DDA is double hexadecyldimethylamines
The abbreviation of base ammonium bromide.Usually about 1 microgram of every dosage is to about 5000 micrograms.Its usage amount also can be for about 1 microgram of every dosage extremely
About 4000 micrograms, about 1 microgram of about 1 microgram of every dosage to about 3000 micrograms, about 1 microgram of every dosage to about 2000 micrograms and every dosage
To about 1000 micrograms.Its usage amount also can for about 5 microgram of every dosage to about 750 micrograms, about 5 microgram of every dosage to about 500 micrograms,
About 5 microgram of every dosage to about 200 micrograms, about 5 microgram of every dosage to about 100 micrograms, about 15 microgram of every dosage to about 100 micrograms and
About 30 microgram of every dosage is to about 75 micrograms.
Preferably, the vaccine composition further comprises pharmaceutically acceptable carrier, described pharmaceutically to connect
The carrier received includes any available solvent, decentralized medium, stabilizer, isotonic agent, diluent or preservative.
" pharmaceutically acceptable carrier includes any solvent for use, isotonic agent, decentralized medium, stabilizer, dilution to term
Agent, preservative.If diluent includes but is not limited to water, phosphate buffer solution, salt water, ethyl alcohol, glycerol, isotonic agent includes but not
It is limited to sodium chloride, dextran, glucose；Stabilizer includes albumen or disodium ethylene diamine tetraacetate, due to albumen can and egg
White enzyme combines, therefore has stabilization.
Antibiotic or preservative can also be contained in composition, include, for example: gentamicin, thimerosal or chloreresol；
It is well known to those skilled in the art to select different classes of antibiotic or preservative.
Preferably, the vaccine composition includes 20 micrograms of Porcine circovirus type 2 ORF2 protein antigen/mL, levamisole hydrochloride 1.8mg/
ML, 10 milligrams/mL and DDA100 of carbomer microgram/mL；Or the vaccine composition includes Porcine circovirus type 2 ORF2 protein 20ug/mL,
Levamisole hydrochloride 1.8mg/mL, carbomer 2mg/mL and DDA50 microgram/mL；Or the vaccine composition includes
Porcine circovirus type 2 ORF2 protein 60 μ g/mL, levamisole hydrochloride 1.8mg/mL, carbomer 2mg/mL and DDA100 microgram/mL.
As a preferred embodiment of the present invention, 20 micrograms of circovirus subunit antigen/dosage, the left-handed miaow of hydrochloric acid
Azoles 1.8mg/ dosage (is equivalent to 1.5 milligrams/dosage of levamisol), 10 milligrams/dosage of carbomer, DDA100 microgram/dosage.
Circovirus subunit antigen 20ug/ dosage, levamisole hydrochloride 1.8mg/ dosage, carbomer 2mg/ dosage,
DDA50 microgram/dosage.
The preferred 6.9-7.5 of PH of the invention.
Another object of the present invention is to provide a kind of methods for preparing the vaccine composition, which comprises
(1) the circovurus type 2 antigen of immune amount is added in carrier；
(2) levamisol or derivatives thereof is added in the circovurus type 2 antigen.
The preparation method of vaccine composition of the present invention, includes the following steps:
A the composition of antigenic component) is prepared in buffer；
B) described levamisol or derivatives thereof is added in step A；
C) DDA is added in the composition of step B；
D) polymer is added in the composition of step C.
A further object of the present invention is to provide the vaccine compositions to treat and prevent pig circular ring virus correlation in preparation
Application in the drug of disease.
From the above, it can be seen that the vaccine adjuvant composition for treating or preventing pig infectious diseases of the invention is at least
Following advantages:
1. vaccine immunity protection can be improved, be free from side effects: subsequent embodiment can be seen that this hair through the invention
Bright vaccine adjuvant can increase substantially the immune efficacy of vaccine, be significantly better than and adjuvant group is not added and only adds carbomer group.
2. the diluent that vaccine of the invention may be used as the live virus antigen of freeze-drying.
3. proving through long-term experiment, vaccine composition of the invention has stability.
Specific embodiment
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with description more
It is clear.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
It should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form carry out
Modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Dosage used in the present embodiment is also also known as " head part " at " immune amount ", refers to that offer is directed to PCV-2 immunizing dose,
It depends primarily on following factor: being before species, kind, age, weight size, health status and the animal of immunized animal
The no vaccine for receiving the same virus of confrontation.
" TCID50 " refers to " tissue culture infection dose ", and is defined as the cell through being inoculated with of 50% given batch of infection
Required viral dilution amount when culture.A variety of methods can be used to be made to calculate in TCID50, including this specification
Spearman-Kappa method (Spearman-Karber method).Spearman-Kappa method retouches B.W.Mahy&
H.O.Kangro, Virology Methods Manual, p.25-46 (1996).
Heretofore described " PBS " refers to the English contracting of phosphate buffer (Phosphate Buffer Saline)
It writes, is the PBS of the pH7.4 of 0.01mM used in the present invention, is prepared by described in " molecular cloning " third edition.
1 levamisole hydrochloride solution of embodiment
By levamisole hydrochloride, dissolution in deionized water, is prepared into the stock solution of 100 mg/mls, micro- with 0.2
Rice filter filtering.
Embodiment 2.DDA solution
By GERBU Adjuvant 100 (DDA；Fluka Analytical) it dissolves in ethanol, 15 milli of preparation
The stock solution of grams per milliliter, with 0.2 zut filter DDA stock solution.
3. carbomer solution of embodiment
In deionized water by carbomer dissolution, the stock solution of 500 mg/mls is prepared.
Embodiment 4.DDA/ carbomer solution
DDA stock solution is prepared according to embodiment 2.The carbomer stock solution of 500 mg/mls is prepared according to embodiment 3, it will
Solution is mixed to obtain required ultimate density.
5. levamisols of embodiment/carbomer solution
It is required to obtain by carbomer solution prepared by embodiment 3 is added in levamisol solution prepared by embodiment 1
Ultimate density, the pH value of the solution is adjusted with NaOH or HCl with obtain required final pH (usually 6.9 to 7.5 range
It is interior).
6 levamisols of embodiment/DDA/ carbomer solution
The DDA solution that will be slowly added to embodiment 2 in levamisole hydrochloride solution prepared by embodiment l and prepare, then plus
The carbomer solution for entering the preparation of embodiment 3 is added to obtain required ultimate density.The pH value of the solution is adjusted with NaOH or HCl
To obtain required final pH (usually in the range of 6.9 to 7.5).
Embodiment 7: this test generates neutralizing antibody facilitation to Porcine circovirus type 2 ORF2 protein for verifying levamisol adjuvant
1. according to the amount of table 1 Porcine circovirus type 2 ORF2 protein is added, then in the preparation of vaccine in the phosphate buffer of pH=7.4
The mixed solution of levamisol carbomer is added, is settled to volume with the phosphate-buffered of pH=7.4.
Table 1: composition matches tabulation
2. pig body neutralizing antibody is tested
By the PCV2 negative healthy piglet of 35 30 ages in days or so, it is randomly divided into 7 groups, every group 5.By above-mentioned 6 kinds of vaccines
The 1st~6 group of piglet, 1mL/ head are injected respectively；The physiological saline of 7th group of injection equivalent is as blank control group.It surveys within 4 weeks after immune
Its fixed neutralize antibody titers.
The detection method of neutralizing antibody is as follows: different dilution inactivated serums are mixed with virus, and 37 DEG C of water-baths act on 1h, are taken out
The PK-15 cell for having covered with cell monolayer, every 100 μ L of hole are added afterwards, each serum dilution is inoculated with 4 holes, concurrently sets and only connect
Positive control wells, normal cell negative control hole and the negative serum control hole of kind virus PCV2.Virus is final concentration of
1000TCID50/mL.It is handled afterwards with the D- Glucosamine of 300mmol/L for 24 hours, is then further cultured for 48h, is immunized indirectly glimmering
Phototesting.With the inverse of the serum highest dilution of reduction 70% or more fluorescence for neutralize antibody titers.
2. neutralizing antibody is horizontal after piglet
1-4 group neutralize antibody titers are between 1:220~1:240.Difference is not significant between group and group, and 5 groups, 6 groups are 1:
Difference is not significant between two groups of 120~1:130, and 1-4 group is with 5-6 group significant difference.
The neutralizing antibody of each test group of table 2
| Group | 1 | 2 | 3 | 4 | 5 | 6 |
| Neutralizing antibody | 1:240 | 1:220 | 1:240 | 1:230 | 1:130 | 1:120 |
Test the results show that the concentration of levamisol generates in a certain range is immunized its humidification, and it is not bright
Aobvious dose-effect relationship, beyond doses instead without immunological enhancement.
Embodiment 8: the Immunization test of composition
1. the preparation of vaccine
According to the amount of table 3, Porcine circovirus type 2 ORF2 protein is added in the phosphate buffer of pH=7.4, left-handed miaow is then added
The mixed solution of azoles, DDA carbomer is settled to volume with the phosphate buffer of pH=7.4.
Table 3: composition matches tabulation
Pig body protest test
By the PCV2 negative healthy piglet of 30 30 ages in days or so, it is randomly divided into 6 groups, every group 5.By above-mentioned 5 kinds of vaccines
The 1st~5 group of piglet, 1mL/ head are injected respectively；The physiological saline of 6th group of injection equivalent is as blank control group.It surveys within 4 weeks after immune
Fixed its neutralize antibody titers PCV attacks poison: 35d after m, each test group and control group piglet respectively use PCV2SH plants (to contain 106.0TCID50/
Ml) collunarium 1ml/, intramuscular injection 2ml/ head, attack after poison the 4th, 7, respectively the two sides oxter in every first tap poison pig and two sides stern
Portion's keyhole hemocyanin (KLH/ICFA, 0.5mg/ml) that totally 4 points emulsify all pig inoculations incomplete Freund's adjuvant,
Each point inoculation 1ml(4ml/), at the same intraperitoneal inoculation thioglycollate medium, 10ml/ head；Attack after poison the 11st, 19 again
Intraperitoneal inoculation thioglycollate medium, 10ml/ head.It is observed continuously 25 after attacking poison, slaughters, cut open after weighing on the 25th after attacking poison
Inspection.Determined that Temperature changing situation is shown in Table 3, vaccine A, vaccine according to body temperature, opposite daily gain and virus antigen detection result
There is not body temperature and increase phenomenon in B and vaccine C immune group piglet, and vaccine D and vaccine E only have individual pigs and transient body occur
Temperature increases phenomenon, and body temperature restores normal after increasing one day quickly, without other clinical symptoms；Control group pig attacks all pigs after poison
Body temperature increases 40.5 DEG C or more, continues 3~5 days, and appetite stimulator, spirit are depressed, coat is thick disorderly, the thin and speed of growth slows down.
It falls ill after attacking poison and protection situation is shown in Table 4,5.Vaccine A, B, C, which are immunized piglet and attack, does not occur obvious clinical condition after poison
Shape does not observe that specific pathologies change, and antigen PCR detection is feminine gender, and protecting effect is up to 5/5；The immune son of vaccine D, vaccine E
After pig attacks poison, there is a morbidity of 1 pig, protecting effect is up to 4/5.And control group piglet all falls ill, clinical symptoms, pathological change is bright
Aobvious, cause of disease PCR detection is the positive.But terminate to poison observation is attacked, vaccine A, vaccine B and vaccine C vaccine D are immunized piglet and are averaged day
Weight gain is without significant difference, and compared with the control group, each vaccine group piglet average daily gain is extremely significant to be higher than control group.
Table 4PCV2 attacks the number of days that each group test temperature of pig body surpasses 40.5 DEG C after poison and compares
The morbidity of each group experimental animal determines result after table 5PCV attacks poison
PCV attacks after poison any 2 met in following 3, can be judged to fall ill.
A. clinical symptoms: piglet body temperature increases (>=40 DEG C), should at least continue 3, and it is heavy obvious appetite stimulator, spirit occur
Strongly fragrant, thick random, the thin and speed of growth of coat slows down；
B. pathological change: groin and lymphoglandulae tracheales oedema, lungs Mild edema, kidney turn to be yellow or have a spotty necrosis.
Histologic lesion is that lymph has obvious lymphocyte intrusion, or has multinucleate giant cell；
C. viral diagnosis: lymph node tissue is detected with PCR, detects PCV2.
Table 6 is immunized after piglet PCV attacks poison and protects situation
Neutralizing antibody is horizontal after piglet
The neutralizing antibody level of each test group is shown in Table 7, and A-C group neutralize antibody titers are in 1:280~1 as seen from the results in Table 7:
Between 300.Difference is not significant between group and group, and E group is 1:130, and with A-C group significant difference, D group is lower than A-C group neutralizing antibody
Level, but it is significantly higher than E group.
The neutralizing antibody of each test group of table 7
| Group | A | B | C | D | E |
| Neutralizing antibody | 1:300 | 1:280 | 1:280 | 1:220 | 1:130 |
9 vaccine consistency check of embodiment
The preparation of inactivated virus vaccine: it is entirely sick that PCV2 is added in the phosphate buffer of pH=7.4 according to the amount of table 8
Then malicious antigen is added the mixed solution of levamisol, DDA, carbomer, is settled to volume with the phosphate-buffered of pH=7.4.
The preparation of table 8, PCV2 inactivated virus vaccine
Take porcine reproductive and respiratory syndrome live vaccine (JXA1-R plants) (Pulaike Biological Engineering Co., Ltd., lot number
1202031), with embodiment 8 prepare vaccine A~D and inactivated virus vaccine and refer to dilution (sterile water for injection),
According to operation instruction, part/bottle (2ml) of inactivated vaccine part 1 is restored.20 ± 3 DEG C of placement 2h breed according to pig and breathe synthesis
Live vaccine titration method is levied to examine.By compared with reference dilution, assessing dilution to be checked to the shadow of virus activity component
It rings, the titration results of virus component such as table 9, difference is no more than the Europe 0.7log10(live vaccine standard).Epidemic disease of the invention
Seedling may be used as the diluent of the live virus antigen of freeze-drying.
9 vaccine consistency check result of table
Embodiment 10PCV2 subunit vaccine prevents and treats the effect of PCV2 infection
1. the preparation of vaccine
According to the amount of table 10, Porcine circovirus type 2 ORF2 protein is added in the phosphate buffer of pH=7.4, left-handed miaow is then added
The mixed solution of azoles, DDA carbomer is settled to volume with the phosphate-buffered of pH=7.4.
10 composition of table matches tabulation
The present embodiment tests the effect of 5 kinds of PCV2 candidate vaccines, the effect after further determined contact PCV2 velogen strain
Rate parameter.35 nascent 9-14 age in days piggys for not eating colostrum are randomly divided into 7 groups, every group 5 of same size.1st group of neck
Portion's intramuscular injection PCV2 subunit vaccine V1(60 μ g/ml) 1ml, the 2nd group of musculi colli injection PCV2 subunit vaccine V2(30 μ
G/ml) 1ml, the 3rd group of musculi colli inject PCV2 subunit vaccine V3(10 μ g/ml) 1ml, the 4th group of musculi colli injection PCV2
Subunit vaccine V4(5 μ g/ml) 1ml, the 5th group of musculi colli injection PCV2 subunit vaccine V5(2 μ g/ml) 1ml, the 6th group is not
Immune, as attacking malicious control group, the 7th group not immune not to attack poison, as blank control group.Immune preceding and immune rear 7d, 14d,
21d, 28d respectively take a blood sample, and separate serum.25 days after immune, the totally 4 point inoculations of two sides oxter and two sides buttocks are distinguished to all pigs
The keyhole hemocyanin (KLH/ICFA, 0.5mg/ml) emulsified with incomplete Freund's adjuvant, each point inoculation 1ml(4ml/
Head).It weighs within 28 days after immune, attack poison, the 1st, 2,3,4,5,6,7,8 group is respectively used PCV2HL plants (to contain 106.0TCID50/ ml) collunarium
2.5ml, intramuscular injection 2.5ml.It attacks after poison the 4th, 7, to all pigs, totally 4 points are connect for two sides oxter and two sides buttocks respectively again
The keyhole hemocyanin (KLH/ICFA, 0.5mg/ml) that kind is emulsified with incomplete Freund's adjuvant, each point inoculation 1ml(4ml/
Head).It takes a blood sample to all pigs within 7th, 14,21,28 after attacking poison.It is observed continuously 28 after attacking poison, daily measurement body temperature and record face
Bed symptom, weighed on 28th, cutd open and kill (the 1st group and the 8th group leave not kill continue to raise), observation pathological change simultaneously takes fresh group
It knits (tonsillotome, lungs, lymphonodi mesenterici, tracheobronchial lymph nodes and inguinal lymph nodes) portion and sets -20 preservations, it is a
It is fixed with 10% formalin.Carry out PCV2PCR detection (regular-PCR and glimmering respectively to flesh tissue and formalin-fixed tissue
Fluorescent Quantitative PCR detection) and IHC detection, PCV2PCR detection (regular-PCR and fluorescence quantitative PCR detection) is carried out to the serum of acquisition
With PCV2 antibody test (ELISA and IFA).Protecting effect is assessed according to Gain weight and PCV2 antigen testing result.Table 11
List the Overall study design of the present embodiment.
The grouping of 11 piglet of table and processing
As a result
Average daily increased weight (ADWG) the results are shown in Table the 12, the 7th group as it is not immune do not attack malicious negative control group its
ADWG highest (1.07+0.20kg/ days), followed by the 1st of immune vaccine V1 the group (0.98+0.22kg/ days), minimum is unavoidably
6th group (0.48+0.30kg/ days) of epidemic disease, remaining different antigenic content vaccine immunity group ADWG difference are little.
Average daily increased weight (ADWG) result compares between 12 groups of table
PCV2 results of serological detection is shown in Table 13,14.In immune all serum on the 0th be all PCV2 antigen and antibody is yin
Property, be immunized after the 14th day, the group potency highest of immune vaccine V2, range is 256~640, potency it is minimum be nonimmune
6th group and the 7th group, the group PCV2 antibody titer difference that different antigenic contents are immunized in remaining is little.28th day after immune, it is immunized
The group potency highest of vaccine V2, range is 6400~25600, potency it is minimum be nonimmune 6th group and the 7th group, remaining
The group PCV2 antibody titer difference that different antigenic contents are immunized is little.It attacks after poison the 14th, the 7th group of PCV2 antigen is detected as yin
Property, the 6th group of PCV2 antigen detected level highest is 2.15 × 106Copy/ml, remaining group differences are little.It attacks after poison the 28th,
7th group of PCV2 antigen is detected as feminine gender, and the 6th group of PCV2 antigen detected level highest is 5.49 × 109Copy/ml, remaining group are poor
It is different little.
PCV2 antigen testing result compares (fluorescent PCR) between 13. groups of table
PCV2 antibody test result (average value) compares (ELISA) between 14 groups of table
There are not any apparent clinical symptoms in all pigs before attacking poison, and continuous body temperature occurs in the 6th group of pig after attacking poison
More than 40 DEG C phenomenons, and there is 1 death, it is short of breath before dead, continuous high fever, remaining group piglet all goes well.It cuts open and kills
PCV2 antigen is detected by immunohistochemistry afterwards, the 6th group of piglet internal organs recall rate is 100%, and the 7th group of recall rate is 0%, remaining group
Recall rate is 0%~20%.Detailed results are shown in Table 15.
PCV2 clinical symptoms and dissect symptom compare between 15. groups of table
Embodiment 11
The present embodiment studies the stability of vaccine composition of the present invention.
5 groups of vaccine V1~V5 prepared by embodiment 10 are placed in 2~8 DEG C of preservations 1 month, 6 months, 12 months, 18 months, 24
A month and 27 months, physical behavior supervision is carried out after taking-up respectively, is occurred without precipitating, to the measurement of the immune efficacy of piglet with
Indifference when just preparing for 0 day.
Since DDA is not soluble in water, and levamisol is unstable in neutral and alkaline conditions, is easy to decompose and precipitate, but
Composition surprisingly of the invention has stability, saves 1 month, 6 months, 12 months, 18 months, 24 months and 27
A month, physical behavior supervision is carried out after taking-up respectively, is occurred without precipitating, but only adds DDA and levamisol group process for preparation base
Originally it cannot dissolve.
It is proved through long-term experiment, 5 groups of vaccine compositions of V1~V5 prepared by the embodiment of the present invention 10 have stability.
The above is only the preferred embodiment of the present invention, not does limitation in any form to the present invention, though
So the present invention is disclosed above with preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this profession
Member, in the range of not departing from technical solution of the present invention, when the technology contents using the disclosure above make a little change or repair
Decorations are the equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, technology according to the present invention are real
Matter any simple modification, equivalent change and modification to the above embodiments, still fall within the range of technical solution of the present invention
It is interior.
Claims (3)
1. a kind of vaccine composition, the vaccine composition includes the circovurus type 2 antigen PCV2 ORF2 2~10 of immune amount
μ g/ml and levamisol or derivatives thereof, polymer, DDA；Wherein, levamisol derivative be levamisol hydrochloride or
Phosphate, the content of described levamisol or derivatives thereof are 1.5mg/ml milligrams/mL；The polymer is carbomer；It is described
Polymer content is 2mg/ml, and DDA content is 100 μ g/ml.
2. vaccine composition according to claim 1, wherein the vaccine composition further comprises that can pharmaceutically connect
The carrier received, the pharmaceutically acceptable carrier includes any available solvent, decentralized medium, stabilizer, isotonic agent, dilute
Release agent or preservative.
3. the medicine that any one of claim 1~2 vaccine composition treats and prevents porcine circovirus associated diseases in preparation
Application in object.
Priority Applications (1)
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| CN101389350A (en) * | 2005-12-29 | 2009-03-18 | 贝林格尔.英格海姆维特梅迪卡有限公司 | Multivalent pcv2 immunogenic compositions and methods of producing such compositions |
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| CN101277717A (en) * | 2005-09-09 | 2008-10-01 | 英特威国际有限公司 | PCV-2 vaccine |
| CN101389350A (en) * | 2005-12-29 | 2009-03-18 | 贝林格尔.英格海姆维特梅迪卡有限公司 | Multivalent pcv2 immunogenic compositions and methods of producing such compositions |
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